Mass MALDI

Fr. match Calc. mass MALDI Obs. mass (m/z) LSIMS Obs. mass (m/z)... 

Figure 5. Methods employed for identifiying and characterizing proteins separated by 2DE. Abbreviations used 2DE, two-dimensional electrophoresis ESI, electrospray ionization HPLC, high performance liquid chromatography IR, infrared M, relative molecular mass MALDI, matrix-assisted laser desorption ionization MS, mass spectrometry MS/MS tandem mass spectrometry p/, isoelectric point PSD, post-source decay.

Traditional methods to generate peptide maps involve fractionation of complex mixtures of peptides in a protein digest either with one-dimensional SDS-PAGE or RP-HPLC [28,29]. The mass spectrometry peptide-mapping protocol, in principle, is similar to these techniques, but it provides an added dimension of structure-specific data (i.e., the molecular mass). MALDI-MS [30,31], ESl-MS [32], LC/ESI-MS [33], and CE/ESI-MS [34] have currently replaced the traditional biochemical approaches. MALDI allows the direct analysis of unfractionated protein digests. The commonly used matrices are sinapinic acid, a-cyano-4-hydroxy cinnamic acid (a-CHCA), and 2,5-dihydroxybenzoic acid (DHB). 

Gasilova, N. and Nazabal, A. (2012) Monitoring ligand modulation of protein-protein interactions by chemical cross-linking and high-mass MALDI mass spectrometry. Methods Mol Biol, 803, 219-229. 

Figure 10.1, High-mass MALDI spectrum recorded using a superconducting tunnel junction detector. In addition to the mass spectrum (a), the detector records ion kinetic energies (b), enabling one to distinguish multiply charged ions and metastables.

Figure 15.3. Sandwich immunocomplex analyzed by high-mass MALDI mass spectrometry A complex formed by two monoclonal antibodies (6H4 and 3B8) against the bovine prion protein (bPrP) has been cross-linked and analyzed by high-mass MALDI TOP mass spectrometry. The specific protein complex [6H4 bPrP bPrP-3B8] is...

Nazabal, A. Schlumberger, M. Hardt, W. Zenobi, R. Super-shifting A new method for the analysis of protein complexes using high-mass Maldi Tof mass spectrometry. In Proceedings of the 54th Conference of the American Society for Mass Spectrometry, May 28-June, 1, 2006, Seattle, WA 2006. 

A connnon feature of all mass spectrometers is the need to generate ions. Over the years a variety of ion sources have been developed. The physical chemistry and chemical physics communities have generally worked on gaseous and/or relatively volatile samples and thus have relied extensively on the two traditional ionization methods, electron ionization (El) and photoionization (PI). Other ionization sources, developed principally for analytical work, have recently started to be used in physical chemistry research. These include fast-atom bombardment (FAB), matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ES). 

Other methods of sample introduction that are commonly coupled to TOP mass spectrometers are MALDI, SIMS/PAB and molecular beams (see section (Bl.7.2)). In many ways, the ablation of sample from a surface simplifies the TOP mass spectrometer since all ions originate in a narrow space above the sample surface. 

Until about the 1990s, visible light played little intrinsic part in the development of mainstream mass spectrometry for analysis, but, more recently, lasers have become very important as ionization and ablation sources, particularly for polar organic substances (matrix-assisted laser desorption ionization, MALDI) and intractable solids (isotope analysis), respectively. 

Modern commercial lasers can produce intense beams of monochromatic, coherent radiation. The whole of the UV/visible/IR spectral range is accessible by suitable choice of laser. In mass spectrometry, this light can be used to cause ablation, direct ionization, and indirect ionization (MALDI). Ablation (often together with a secondary ionization mode) and MALDI are particularly important for examining complex, intractable solids and large polar biomolecules, respectively. 

For solids, there is now a very wide range of inlet and ionization opportunities, so most types of solids can be examined, either neat or in solution. However, the inlet/ionization methods are often not simply interchangeable, even if they use the same mass analyzer. Thus a direct-insertion probe will normally be used with El or Cl (and desorption chemical ionization, DCl) methods of ionization. An LC is used with ES or APCI for solutions, and nebulizers can be used with plasma torches for other solutions. MALDI or laser ablation are used for direct analysis of solids. 

When mass spectrometry was first used as a routine analytical tool, El was the only commercial ion source. As needs have increased, more ionization methods have appeared. Many different types of ionization source have been described, and several of these have been produced commercially. The present situation is such that there is now only a limited range of ion sources. For vacuum ion sources, El is still widely used, frequently in conjunction with Cl. For atmospheric pressure ion sources, the most frequently used are ES, APCI, MALDI (lasers), and plasma torches. 

Mass-Analyzed Laser Desorption Ionization (MALDI)... 

Laser-desorption mass spectrometry (LDMS) or matrix-assisted laser desorption ionization (MALDI) coupled to a time-of-flight analyzer produces protonated or deprotonated molecular ion clusters for peptides and proteins up to masses of several thousand. 

Peptides and proteins can be analyzed by mass spectrometry. Molecular mass information can be obtained particularly well by MALDI and ESI. 

MALDI = matrix assisted laser desorption, ftms = Fourier transform mass spectrometry TOF = time of flight. 

Matrix-assisted laser desorption/ionization (MALDI) is widely used for the detection of organic molecules. One of the limitations of the method is a strong matrix background in low-mass (up to 500-700 Da) range. In present work an alternative approach based on the application of rough matrix-less surfaces and known as surface-assisted laser desoi ption/ionization (SALDI), has been applied. 

Figure 12.9 MALDI-TOF mass spectrum of chicken egg-white lysozyme. The peak at 14,307.7578 daltons (amu) is due to the monoprotonated protein, M+H+, and that at 28,614.2188 daltons is due to an impurity formed by dimerization of the protein. Other peaks are various protonated species, M+H rH ...

With the identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amino acids are linked together. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in Section 12.4. Also in common use is a chemical method of peptide sequencing called the Edman degradation. 

MALDI (Section 12.4) Matrix-assisted laser desorption ionization a mild method for ionizing a molecule so that fragmentation is minimized during mass spectrometry. 

Major groove (DNA), 1104-1105 Malate, from fumarate, 221-222 MALDI-TOF mass spectrometry, 417-418... 

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